我们感谢Moustafa Saleh和Jay Tourigny对手稿的批判性阅读，Steven Hahn和Steven Henikoff在开发ChEC-seq期间的指导和支持及其在Mediator综合体中的应用。 SG由NIH授权R01GM053451和R01GM075114支持，GEZ由印第安纳大学创业基金支持。

酵母菌的生成<ol><li> 产生带有MNase标记的感兴趣因子的酵母菌株。 <ol><li> PCR使用指定的反应混合物（ 表2 ）和循环条件（ 表3 ）从所需载体（ 表1 ）扩增MNase标记盒。混合5μLPCR反应和1μL6X DNA加载染料。在0.8V的琼脂糖凝胶上以120V的速度运行每个PCR等分试样40分钟。预期产品大小为〜2.3 kb。 </li><li>使用标准乙酸锂转化22将放大的标记盒转化为选择的菌株，并进行选择。 </li><li>验证标签盒与菌落PCR的正确整合。 <ol><li>为指定的反应混合物（ 表4 ）准备待筛选的菌落数。保持在冰上直到需要。 </li><li>选择每个殖民地的一部分是tes使用无菌牙签或移液管吸头进入PCR管的底部。 </li><li>微波挑选的殖民地在高功率1分钟。 </li><li>向每个微波菌落加入50μLPCR混合物（ 表4 ），上下移液管混匀。使用指定的循环条件进行PCR（ 表5 ）。 </li><li>在每个PCR管中直接混合50μLPCR反应和10μL6X DNA加载染料。在120℃下，在1％琼脂糖凝胶上运行10μL每个样品40分钟。预计产品大小约700 bp。 注意:如果需要，通过蛋白质印迹验证MNase融合蛋白的适当表达。预期标记蛋白的分子量约为20.6千道尔顿。 </li></ol></li></ol></li><li> 通过基因克隆将目的基因的启动子与pGZ136（表1）和转化和选择同步进行克隆，产生免费的MNase菌株，如步骤1.1.2所示。 <ol><li> Alternati将感兴趣的基因的启动子和3xFLAG-MNase-SV40 NLS组装到质粒载体中，如步骤1.1.2所示进行转化和选择，并在合适的选择条件下生长菌株以维持质粒。 </li></ol></li></ol> ChEC <ol><li>在ChEC实验前一天的下午/晚上，用含有MNase标记因子的菌株的单个菌落接种3mL酵母蛋白胨 - 葡萄糖（YPD）或合适的选择培养基。将该培养物孵育过夜（180RPM摇动，30℃）。 </li><li>在ChEC实验当天的早晨，将过夜培养物稀释到50mL的培养基中至600nm的光密度（OD 600 ）为0.2-0.3。孵育该培养物（180RPM摇动，30℃），直至达到0.5-0.7的OD 600 。当培养物接近适当的OD 600时，将热块或水浴置于30℃，开始解冻缓冲液A添加剂（ Ta6）。 </li><li>每个培养物准备5 mL缓冲液A加添加剂（ 表6 ）。在程序中保持缓冲液A在RT。 </li><li>准备收集每个时间点的含有90μL终止液（ 表7 ）和10μL10％SDS的微量离心管。对于分析的每个新因子，在0秒，30秒，1分钟，2.5分钟，5分钟和10分钟取样。 </li><li>将滗析培养物倒入50mL锥形管中，并以1,500×g和室温（RT）离心1分钟。 </li><li>将细胞重新悬浮在1mL缓冲液A中，并将悬浮的细胞转移到微量离心管中。将颗粒在1,500×g和RT中重悬浮于微量离心机中30秒。 </li><li>吸出上清液，并通过上下移液将细胞彻底重悬于1mL缓冲液A中。将颗粒在1,500×g和RT中重悬浮于微量离心机中30秒。重复此步骤一次。 </li><li>在570μL缓冲液A中彻底重悬细胞。加入30μL的2％洋地黄皂苷（0.1％终浓度）以促进细胞的透化，并反转混合。 </li><li>在30℃的热封或水浴中使细胞渗透5分钟。 </li><li>上下反应以确保细胞的均匀分布。去除100μL等分的透化细胞作为阴性对照至含有终止溶液和SDS的微量离心管（步骤2.4）并短暂涡旋混合。 </li><li>向透化细胞中加入1.1μL1M CaCl 2 （2mM终浓度）以激活MNase并快速反转数次或短暂涡旋混合。立即在30°C返回混合反应并启动定时器。 </li><li>在每个时间点收集，上下移动反应以确保细胞的均匀分布，然后将100μL等渗透细胞的细胞除去含有终止液和SDS的微量离心管，并短暂涡旋混合。对于所分析的每个新因子，取0 s（不含CaCl 2 ），30 s，1分钟，2.5分钟，5分钟和10分钟时间点。 注意:可以在较低温度下进行在30℃快速切割DNA的因子的ChEC实验，以减缓MNase切割动力学。 </li><li>收集所有时间点后，向每个样品加入4μL20 mg / mL蛋白酶K，短暂旋转混匀，55℃孵育30 min。 </li><li>向每个样品中加入200μL25:24:1苯酚:氯仿:异戊醇，剧烈旋转混合，并在微量离心机中以最大速度和RT离心5分钟。 </li><li>去除每个水相（〜150μL）到一个新的管。加入30μg糖原和500μL100％乙醇。大量涡旋混合并在干冰上沉淀10分钟，或直到溶液粘稠。 </li><li>在微量离心机中以最大速度和4℃沉淀DNA沉淀10分钟。 </li><li>倾析上清液，并用1mL RT 70％乙醇洗涤沉淀。 </li><li>倾倒乙醇，通过倒置去除剩余物d轻轻地将管放在纸巾上。使用台式离心机简单旋转样品，并使用移液管去除残留的乙醇，注意不要打扰沉淀。空气干燥颗粒在室温下5分钟。 </li><li>当颗粒干燥时，制成主混合物，将干燥的沉淀物重悬于29μLTris，pH 8.0 +1μL的10mg / mL核糖核酸酶A中。在37℃下消化RNA 20分钟。 </li><li>将1μL6X DNA负载染料与5μL每个样品混合，并在120V下在1.5％琼脂糖凝胶上运行40分钟。 </li></ol> 3.尺寸选择注意:大小选择的目标是从待测序的样品中除去基因组DNA的多千碱基片段，并富集〜150bp的片段（大约为核小体DNA的大小）或更小。在测序数据中，富集了400bp的片段，在核小体DNA（〜150bp）的大小周围具有显着的峰值，并且亚核核糖体片段的峰或宽分布。 &lt;OL&gt; <li>向每个样品中加入75μL固相可逆固定（SPRI）珠粒在聚乙二醇（PEG）溶液23中 ，并在上下移液10次以混合。在室温孵育珠:DNA混合物5分钟。 </li><li>在SPRI珠孵育期间，准备每个样品含有96μL10 mM Tris，pH 8.0和4μL5 M NaCl的微量离心管。 </li><li>将管放在磁性架中以收集管侧。允许珠在管的一侧收集2分钟。 </li><li>将每个上清液转移到含有Tris和NaCl的新管中（步骤3.2）。 </li><li>向每个样品中加入200μL25:24:1苯酚:氯仿:异戊醇，涡旋混合，并在微量离心机中以最大速度和RT离心5分钟。 </li><li>将每个水相去除一个新的管。加入30μg糖原和500μL100％乙醇。在干冰上沉淀DNA 10分钟或溶液粘稠。 </li><li>颗粒preci在微量离心机中以最大速度和4℃排空DNA 10分钟。 </li><li>去除上清液并用1mL 70％乙醇洗涤沉淀。 </li><li>倾倒乙醇，通过翻转并轻轻敲打纸巾上的管，取出其余部分。使用台式离心机简单旋转样品，并使用移液管去除残留的乙醇，注意不要打扰沉淀。空气干燥颗粒在室温下5分钟。 </li><li>将干燥的沉淀物重悬于25μLTris，pH 8.0中。使用高灵敏度测定法确定样品浓度。对于TF ChEC实验，在大小选择后常规回收10-100ng DNA。 </li><li>准备测序库。如前所述，不依赖于大小选择的文库制备方案是期望的，以允许大范围片段大小（25-500bp）的测序。 </li><li>配对模式下的序列库。高质量阅读需要每端至少25个碱基序列映射。 1至2百万次读数为ChEC样品提供足够的覆盖率。 注意:ChEC-seq数据的分析是一个复杂的过程，超出了本文的范围。有关数据分析的详细信息可以在以前的出版物1,21中找到，用于分析的自定义脚本可在GitHub（https://github.com/zentnerlab/chec-seq）上获得。 HOMER 25 （http://homer.salk.edu）也可用于ChEC-seq数据的命令行分析。 </li></ol>

<ol>
	<li>Zentner, G. E., Kasinathan, S., Xin, B., Rohs, R., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChEC-seq+kinetics+discriminates+transcription+factor+binding+sites+by+DNA+sequence+and+shape+in+vivo.">ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo.</a> Nat Commun. 6, 8733 (2015).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Dynamics+of+Enhanceosome+Assembly+in+Embryonic+Stem+Cells.">Single-Molecule Dynamics of Enhanceosome Assembly in Embryonic Stem Cells.</a> Cell. 156 (6), 1274-1285 (2014).</li><li>Teytelman, L., Thurtle, D. M., Rine, J., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+expressed+loci+are+vulnerable+to+misleading+ChIP+localization+of+multiple+unrelated+proteins.">Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins.</a> Proc Natl Acad Sci USA. 110 (46), 18602-18607 (2013).</li><li>Park, D., Lee, Y., Bhupindersingh, G., Iyer, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+Misinterpretable+ChIP-seq+Bias+in+Yeast.">Widespread Misinterpretable ChIP-seq Bias in Yeast.</a> PLoS ONE. 8 (12), 83506 (2013).</li><li>Jain, D., Baldi, S., Zabel, A., Straub, T., Becker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+promoters+give+rise+to+false+positive+'Phantom+Peaks'+in+ChIP-seq+experiments.">Active promoters give rise to false positive 'Phantom Peaks' in ChIP-seq experiments.</a> Nucleic Acids Res. , (2015).</li><li>Krebs, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+transcription+factor+binding+map+accuracy+utilizing+knockout-mouse+models.">Optimization of transcription factor binding map accuracy utilizing knockout-mouse models.</a> Nucleic Acids Res. 42 (21), 13051-13060 (2014).</li><li>Paul, E., Zhu, Z. I., Landsman, D., Morse, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Association+of+Mediator+and+RNA+Polymerase+II+in+Wild-Type+and+Mediator+Mutant+Yeast.">Genome-Wide Association of Mediator and RNA Polymerase II in Wild-Type and Mediator Mutant Yeast.</a> Mol Cell Biol. 35 (1), 331-342 (2015).</li><li>Worsley Hunt, R., Wasserman, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+transcription+factors+motifs+are+a+systemic+component+of+ChIP-seq+datasets.">Non-targeted transcription factors motifs are a systemic component of ChIP-seq datasets.</a> Genome Biol. 15 (7), 412 (2014).</li><li>Vega, V. B., Cheung, E., Palanisamy, N., Sung, W. -. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherent+Signals+in+Sequencing-Based+Chromatin-ImmunoPrecipitation+Control+Libraries.">Inherent Signals in Sequencing-Based Chromatin-ImmunoPrecipitation Control Libraries.</a> PLoS ONE. 4 (4), 5241 (2009).</li><li>Teytelman, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Chromatin+Structures+on+DNA+Processing+for+Genomic+Analyses.">Impact of Chromatin Structures on DNA Processing for Genomic Analyses.</a> PLoS ONE. 4 (8), 6700 (2009).</li><li>Rhee, H. S., Pugh, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Genome-wide+Protein-DNA+Interactions+Detected+at+Single-Nucleotide+Resolution.">Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution.</a> Cell. 147 (6), 1408-1419 (2011).</li><li>He, Q., Johnston, J., Zeitlinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-nexus+enables+improved+detection+of+in+vivo+transcription+factor+binding+footprints.">ChIP-nexus enables improved detection of in vivo transcription factor binding footprints.</a> Nat Biotech. 33 (4), 395-401 (2015).</li><li>Kasinathan, S., Orsi, G. A., Zentner, G. E., Ahmad, K., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+mapping+of+transcription+factor+binding+sites+on+native+chromatin.">High-resolution mapping of transcription factor binding sites on native chromatin.</a> Nat Methods. 11 (2), 203-209 (2014).</li><li>Skene, P. J., Hernandez, A. E., Groudine, M., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nucleosomal+barrier+to+promoter+escape+by+RNA+polymerase+II+is+overcome+by+the+chromatin+remodeler+Chd1.">The nucleosomal barrier to promoter escape by RNA polymerase II is overcome by the chromatin remodeler Chd1.</a> eLife. 3, 02042 (2014).</li><li>Aughey, G. N., Southall, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dam+it's+good!+DamID+profiling+of+protein-DNA+interactions.">Dam it's good! DamID profiling of protein-DNA interactions.</a> Wiley Interdiscip Rev Dev Biol. 5 (1), 25-37 (2016).</li><li>Wang, H., Mayhew, D., Chen, X., Johnston, M., Mitra, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calling+Cards+enable+multiplexed+identification+of+the+genomic+targets+of+DNA-binding+proteins.">Calling Cards enable multiplexed identification of the genomic targets of DNA-binding proteins.</a> Genome Res. 21 (5), 748-755 (2011).</li><li>Schmid, M., Durussel, T., Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIC+and+ChEC:+Genomic+Mapping+of+Chromatin+Proteins.">ChIC and ChEC: Genomic Mapping of Chromatin Proteins.</a> Mol Cell. 16 (1), 147-157 (2004).</li><li>Merz, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actively+transcribed+rRNA+genes+in+S.+cerevisiae+are+organized+in+a+specialized+chromatin+associated+with+the+high-mobility+group+protein+Hmo1+and+are+largely+devoid+of+histone+molecules.">Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules.</a> Genes Dev. 22 (9), 1190-1204 (2008).</li><li>Schmid, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nup-PI:+The+Nucleopore-Promoter+Interaction+of+Genes+in+Yeast.">Nup-PI: The Nucleopore-Promoter Interaction of Genes in Yeast.</a> Mol Cell. 21 (3), 379-391 (2006).</li><li>Dunn, T., Gable, K., Beeler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cellular+Ca2++by+yeast+vacuoles.">Regulation of cellular Ca2+ by yeast vacuoles.</a> J Biol Chem. 269 (10), 7273-7278 (1994).</li><li>Gr&#252;nberg, S., Henikoff, S., Hahn, S., Zentner, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediator+binding+to+UASs+is+broadly+uncoupled+from+transcription+and+cooperative+with+TFIID+recruitment+to+promoters.">Mediator binding to UASs is broadly uncoupled from transcription and cooperative with TFIID recruitment to promoters.</a> The EMBO Journal. 35 (22), 2435-2446 (2016).</li><li>Gietz, R. D., Schiestl, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+yeast+transformation+using+the+LiAc/SS+carrier+DNA/PEG+method.">High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method.</a> Nat. Protocols. 2 (1), 31-34 (2007).</li><li>Rohland, N., Reich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective,+high-throughput+DNA+sequencing+libraries+for+multiplexed+target+capture.">Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture.</a> Genome Res. 22 (5), 939-946 (2012).</li><li>Orsi, G. A., Kasinathan, S., Zentner, G. E., Henikoff, S., Ahmad, K., Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+regulatory+factors+by+immunoprecipitation+from+native+chromatin.">Mapping regulatory factors by immunoprecipitation from native chromatin.</a> Current protocols in molecular biology. 110, 21-25 (2015).</li><li>Heinz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Combinations+of+Lineage-Determining+Transcription+Factors+Prime+cis-Regulatory+Elements+Required+for+Macrophage+and+B+Cell+Identities.">Simple Combinations of Lineage-Determining Transcription Factors Prime cis-Regulatory Elements Required for Macrophage and B Cell Identities.</a> Mol Cell. 38 (4), 576-589 (2010).</li><li>Kungulovski, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+histone+modification-specific+interaction+domains+as+an+alternative+to+antibodies.">Application of histone modification-specific interaction domains as an alternative to antibodies.</a> Genome Res. 24 (11), 1842-1853 (2014).</li><li>Vogel, M. J., Peric-Hupkes, D., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+in+vivo+protein-DNA+interactions+using+DamID+in+mammalian+cells.">Detection of in vivo protein-DNA interactions using DamID in mammalian cells.</a> Nat. Protocols. 2 (6), 1467-1478 (2007).</li><li>Feeser, E. A., Wolberger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+Functional+Studies+of+the+Rap1+C-Terminus+Reveal+Novel+Separation-of-Function+Mutants.">Structural and Functional Studies of the Rap1 C-Terminus Reveal Novel Separation-of-Function Mutants.</a> J Mol Biol. 380 (3), 520-531 (2008).</li><li>Neph, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+expansive+human+regulatory+lexicon+encoded+in+transcription+factor+footprints.">An expansive human regulatory lexicon encoded in transcription factor footprints.</a> Nature. 489 (7414), 83-90 (2012).</li><li>Mountjoy, K. G., Kong, P. L., Taylor, J. A., Willard, D. H., Wilkison, W. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Melanocortin+receptor-mediated+mobilization+of+intracellular+free+calcium+in+HEK293+cells.">Melanocortin receptor-mediated mobilization of intracellular free calcium in HEK293 cells.</a> Physiol Genomics. 5 (1), 11-19 (2001).</li><li>Seurin, D., Lombet, A., Babajko, S., Godeau, F., Ricort, J. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-Like+Growth+Factor+Binding+Proteins+Increase+Intracellular+Calcium+Levels+in+Two+Different+Cell+Lines.">Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines.</a> PLoS ONE. 8 (3), e59323 (2013).</li><li>Grienberger, C., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Calcium+in+Neurons.">Imaging Calcium in Neurons.</a> Neuron. 73 (5), 862-885 (2012).</li><li>Sander, J. D., Joung, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas+systems+for+editing,+regulating+and+targeting+genomes.">CRISPR-Cas systems for editing, regulating and targeting genomes.</a> Nat Biotech. 32 (4), 347-355 (2014).</li><li>Filion, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+Protein+Location+Mapping+Reveals+Five+Principal+Chromatin+Types+in+Drosophila+Cells.">Systematic Protein Location Mapping Reveals Five Principal Chromatin Types in Drosophila Cells.</a> Cell. 143 (2), 212-224 (2010).</li><li>van Bemmel, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Network+Model+of+the+Molecular+Organization+of+Chromatin+in+Drosophila.">A Network Model of the Molecular Organization of Chromatin in Drosophila.</a> Mol Cell. 49 (4), 759-771 (2013).</li><li>Izzo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genomic+Landscape+of+the+Somatic+Linker+Histone+Subtypes+H1.1+to+H1.5+in+Human+Cells.">The Genomic Landscape of the Somatic Linker Histone Subtypes H1.1 to H1.5 in Human Cells.</a> Cell Rep. 3 (6), 2142-2154 (2013).</li></ol>

作者没有什么可以披露的。

我们已经表明，ChEC可以将不同类型的酵母蛋白质映射到染色质上，并且预计它将广泛适用于酵母中TF和其他染色质结合因子的不同家族。 ChEC-seq的优点在于它不需要交联，染色质溶解或抗体。因此，ChEC避免了X-ChIP-seq中可能存在的伪像，例如超级ChIPable伪影3,4和原生ChIP，例如由于不完全的蛋白质溶解引起的假阴性14 。与所有酶融合方法一样，ChEC-seq的主要缺点是融合蛋白的需要。这可以在萌芽酵母中快速实现，但在后生动物中更加费力。 ChEC-seq的另一个限制是它不能按照组蛋白修饰的分析来实现。然而，修饰结合域可以融合到MNase并表达，使得h使用ChEC-seq进行istone修改映射。在这种情况下，使用表位标记的组蛋白修饰结合结构域的ChIP-seq已被用于绘制基因组范围的组蛋白修饰模式26 。 使用免费的MNase控制在确定ChEC-seq结果的特异性方面很重要。免费的MNase菌株在用于所关心的基因的内源启动子的控制下携带用3xFLAG标记的MNase和猿猴病毒40核定位信号（SV40NLS）。它在概念上类似于DamID研究27中使用的未融合的大坝控制，以及由于染色质可及性导致的非特异性切割的控制。作为TF ChEC-seq的免费MNase控制，我们在ura3位点1 整合了由REB1启动子驱动的3xFLAG-MNase-SV40 NLS。对于Mediator ChEC-seq，我们在非积分血浆中表达了在MED8启动子控制下的3xFLAG-MNase-SV40 NLSd载体保持在选择培养基21中 。在两种情况下，观察到游离的MNase几乎没有裂解。因此，其中3xFLAG-MNase-SV40 NLS由相对鲁棒的启动子驱动的单一对照菌株应适用于大多数实验。 在规划ChEC实验时，对感兴趣的蛋白质的结构考虑可能很重要。例如，我们发现在Reb1的C末端附加MNase给出Reb1基序周围的不对称切割模式，而具有N端MNase的Reb1表达产生对称切割模式1 。我们将这些不同的切割模式归结为Reb1的结构。 Reb1的DNA结合域位于其C末端;因此，C-末端被结构化并且接近于DNA，限制了MNase的运动范围并且仅允许C末端切割。相比之下，Abf1的C末端是相对非结构化的，因此可以增加到到达MNase，允许在Abf1结合位点的两侧分裂。在Rap1的另一种酵母GRF的情况下，我们发现缩短C-末端和MNase之间的接头从33到aa足以严重地减少裂解，这可能是由于所提出的Rap1的C-末端部分的距离来自DNA 28 。当尝试映射存在于大复合物中的蛋白质的结合时，这种结构考虑也可能是重要的。对于我们与酵母基因组21结合的Mediator的ChEC-seq分析，我们选择了C末端被结构预测为暴露的亚基，而不是埋在复合物内。在与MNase融合的三个亚基中，没有强力切割DNA，表明空间限制或与其他DNA结合因子的相互作用减弱了DNA切割。 我们发现ChEC-seq检测到酵母GRF Abf1，Rap1和Reb的峰值是4-12倍所有时间点一起被考虑在内的各种ChIP方法1 1 。这些位点可以分为基于MNase切割动力学的两个类别。酵母GRF的ChEC-seq数据分析显示两个时间上不同的结合位点类别1 。第一个称为"快速"，显示最大切割&lt;1分钟加钙后，通常含有强大的匹配已知的共识图案。第二个称为"慢"，花费了几分钟才能达到明显的切割水平，并且耗尽了主题匹配。平均而言，这两个类别的网站在ChIP数据集中均具有丰富的约束力，尽管在这些ChIP研究中它们并不一定被称为峰值。给定因素的大多数网站是缓慢的网站。我们推测，快速位点是高亲和力转录因子结合位点的代表，而慢位点代表了在结合位点扫描过程中瞬时采样的基因座。 ob通过MNase切割的动力学分离的两类结合位点的保守性可以解释许多ChIP-seq数据集中观察到具有良好建立的DNA结合特异性的给定因子的大量峰不包含共有基序29 :甲醛交联，在大多数ChIP方案中进行10-15分钟，可以重复捕获因子与基因组的瞬时或机会交互作用，导致信号通量。实际上，ChIP-exo和活细胞成像的比较表明，情况2 。值得注意的是，我们没有观察到Mediator ChEC-seq信号21的这种戏剧性的时间依赖性降低，调节器ChEC-seq信号在30秒至20分钟之间相对恒定。鉴于这些观察结果，我们建议执行短期ChEC-seq来恢复高信度结合位点。 我们预计ChEC-seq可以适应非酵母系统。未刺激的哺乳动物细胞中游离钙的浓度为50-300nM 30,32，远低于有效的MNase活化的阈值。因此，在后生动物系统中建立ChEC-seq的限制步骤是融合蛋白的产生，其在后生动物中比酵母细胞更费力。然而，基于CRISPR的基因组工程33的出现大大促进了后生动物基因的内源标记，因此这个限制应该很容易被克服。或者，MNase融合蛋白可以以低水平从质粒表达。这种方法在果蝇 34,35和人36细胞中的DamID已经相当成功，因此也可以促进后生动物细胞中的ChEC-seq。

映射转录因子（TF），染色质重组体和其他染色质相关调控因子的结合位点是理解所有基于染色质的过程的关键。虽然染色质免疫沉淀和高通量测序（ChIP-seq）方法已被用于获取对基因组生物学的许多重要见解，但它们具有显着的局限性。我们最近引入了一种替代方法，称为染色质内源性切割和高通量测序（ChEC-seq） 1 ，以克服这些缺点。 最常使用初始甲醛交联步骤（X-ChIP-seq）进行ChIP-seq以保护蛋白质-DNA相互作用。然而，一些最近的研究表明，X-ChIP-seq捕获瞬时或非特异性蛋白质 - DNA相互作用2，3，4，5 ，sup&gt;， 6，7，8 ，导致假阳性结合位点。此外，通常用于在X-ChIP-seq实验中分离染色质的超声处理，优先剪切开放染色质区域，导致片段从这些区域偏向恢复9,10。超声处理还产生片段长度的不均匀混合物，最终限制了结合位点分辨率，尽管加入外切核酸酶消化步骤可以极大地提高分辨率11,12 。来自亲和纯化的天然分离染色质（ORGANIC） 13的基因组的占据区域的本地ChIP方法不使用交联和片段染色质与微球菌核酸酶（MNase），减轻与甲醛交联和超声处理相关的潜在偏差。然而，许多染色质结合蛋白在天然染色质提取所需的相对温和条件下的溶解性差，可能导致动态范围和/或假阴性减少14 。 虽然ChIP-seq的各种迭代最常用于蛋白质-DNA相互作用的全基因组映射，但是还已经实现了基于目的蛋白质融合到各种DNA修饰酶的几种测绘技术。一种这样的方法是DNA腺嘌呤甲基转移酶鉴定（DamID） 15 ，其中感兴趣的染色质结合蛋白遗传融合到Dam，并且该融合在细胞或动物中表达，导致接近蛋白质结合位点的GATC序列的甲基化。 DamID的优点在于它不依赖于免疫沉淀，因此避免交联，抗体或染色质溶解。它也在体内进行 。然而，DamID的分辨率限于千碱基量表，Dam融合蛋白的甲基化活性是组成型的。基于酶融合的第二种方法是Calling Card-seq 16 ，其使用感兴趣因子融合转座酶，引导位点特异性整合转座子。像DamID一样，Calling Card-seq不是基于免疫沉淀，因此具有类似的优点，增加分辨率的附加益处。然而，呼叫卡-seq可能受到转座酶的序列偏差的限制，并且还依赖于接近转座子插入位点的限制性位点的存在。 在Laemmli实验室开发的第三种酶融合方法是染色质内源性切割（ChEC） 17 。在ChEC中，染色质相关蛋白和MNase之间的融合物在细胞中表达，并且通过钙添加来活化MNase，DNA被切割到接近标记的结合位点因子（ 图1 ）。结合Southern印迹，ChEC已被用于表征17,18号酵母中多个单个位点的染色质结构和蛋白结合，并结合低分辨率微阵列分析来探测核孔组分与酵母的相互作用基因组19 。 ChEC提供类似DamID和Calling Card-seq的优点，当通过引物扩展19进行分析时，其分辨率几乎为单碱基对。 ChEC也是可控的:MNase的强力DNA切割取决于加入毫摩尔钙，确保MNase在活酵母细胞中观察到的低游离钙浓度下是无活性的。 以前，我们假设将ChEC与高通量测序（ChEC-seq）相结合将提供TF结合位点的高分辨率图谱。事实上，ChEC-seq生成跨基因组1的出芽酵母总调控因子（GRFs）Abf1，Rap1和Reb1的高分辨率图 。我们还成功地将ChEC-seq应用于模块化Mediator复合体，这是一种保守的，重要的全局转录共激活因子21 ，扩大了ChEC-seq对不直接接触DNA的巨大体细胞复合物的适用性，可能难以通过ChIP-基于方法。 ChEC-seq是独立验证ChIP-seq结果和产生对染色质定位过程调节的新见解的有力方法。在这里，我们提出了一种在芽殖酵母中实施该方法的分步骤方案。

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">dNTPs</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">N0447</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Q5 high-fidelity DNA polymerase</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">M0491L</td>
			<td>Other high-fidelity DNA polymerases, such as Phusion, may be used for cassette amplification.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">TrackIt 1 Kb Plus DNA ladder</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:119px;">10488085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Taq DNA polymerase</td>
			<td style="width:159px;">NEB</td>
			<td style="width:119px;">M0273L</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:307px;">cOmplete Mini EDTA-free protease inhibitor cocktail</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:119px;">11836170001</td>
			<td>It is important that an EDTA-free protease inhibitor mix is used, so as not to inhibit Mnase cleavage by chelation of Ca2+.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">PMSF</td>
			<td style="width:159px;">ACROS Organics</td>
			<td style="width:119px;">AC215740010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Digitonin, High Purity</td>
			<td style="width:159px;">EMD Millipore</td>
			<td style="width:119px;">300410-250MG</td>
			<td>Make a 2% stock by dissolving 20 mg digitonin in 1 mL DMSO with vigorous vortexing.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Proteinase K, 20 mg/mL</td>
			<td style="width:159px;">Invitrogen</td>
			<td style="width:119px;">25530049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">RNase A, 10 mg/mL</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EN0531</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">Ampure XP beads</td>
			<td style="width:159px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td>Ampure-like beads can be generated using a published protocol (ref 24).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:307px;">MagneSphere magnetic rack</td>
			<td style="width:159px;">Promega</td>
			<td style="width:119px;">Z5342</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide mapping of protein-dna interactions with chec-seq in saccharomyces cerevisiae

蛋白质 - DNA相互作用的全基因组映射对于了解基因调控，染色质重塑和其他染色质定位过程至关重要。甲醛交联随后进行染色质免疫沉淀和高通量测序（X-ChIP-seq）已被用于获得许多对基因组生物学的宝贵见解。然而，X-ChIP-seq具有与交联和超声处理有关的显着的局限性。原生ChIP通过省略交联来避免这些缺点，但通常导致染色质结合蛋白的恢复不良。此外，所有基于ChIP的方法均受抗体品质的考虑。用于测定蛋白质-DNA相互作用的酶学方法，其涉及将目的蛋白质融合到DNA修饰酶中，也用于测定蛋白质-DNA相互作用。我们最近将一种这样的方法，染色质内源性切割（ChEC）与高通量测序合并为ChEC-seq。 ChEC-seq依赖于染色质相互融合将微生物核酸酶（MNase）感兴趣的蛋白质在活细胞中的钙存在下产生靶向的DNA切割。 ChEC-seq不是基于免疫沉淀，因此避免了交联，超声，染色质溶解和抗体质量的潜在问题，同时以最小的背景信号提供高分辨率图谱。我们设想ChEC-seq将成为ChIP的强大对手，提供独立的手段来验证ChIP-seq结果并发现对基因组调控的新见解。

在成功的ChEC实验的情况下，通过琼脂糖凝胶电泳分析DNA将显示DNA片段化随时间的钙依赖性增加，如通过涂抹和最终完全消化基因组DNA所指示的。在一些情况下，在扩展消化后观察到类似于传统MNase消化所观察到的条带。 Reb1的ChEC分析就是这种情况，Reb1是结合核小体耗尽区域（NDR）的一般调控因子（ 图2 ）。我们发现，在GRF的情况下，延长的消化导致快速切割的结合位点处的信号丢失1 。理想情况下，将使用显示高分子量涂片（例如Reb1 ChEC的30秒和1分钟时间点）的基因组DNA条带尺寸减小的样品进行测序，以避免时间依赖性信号丢失。 图3 ）。类似地，我们通过Mediator亚基Med8与MNase的融合观察到大量的DNA切割，但不加入MED8启动子驱动的游离MNase ，钙加入后1分钟（ 图3 ）。 为了将Reb1 ChEC-seq数据与ChIP-seq进行比较，我们获得了由ORGANIC 13确定的1,991个Reb1峰的列表。这些峰以基序中心为基点，在基序中点周围的100bp窗口中的每个基本位置的平均片段末端计数。我们观察到切割的显着不对称性，大部分片段末端映射到图案的上游侧（图4 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55836/55836fig1.jpg" /> 图1 :ChEC-seq方法的示意图。染色质相关蛋白（CAP）-MNase融合在酵母细胞中表达。该蛋白质与DNA结合，但由于细胞核中游离钙非常低，因此不会产生高于背景水平的切割。在用白细胞渗透细胞和加入毫摩尔钙时，与基因组结合的CAP-MNase融合物切割DNA，释放小片段。然后将这些片段纯化，测序并映射回基因组，从而产生CAP-MNase融合结合位点附近的片段末端的峰。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55836/55836fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图2 :来自Reb1ChEC实验的DNA的琼脂糖凝胶电泳。在大小选择之前，在1.5％TAE-琼脂糖凝胶上分析来自每个ChEC时间点的5μL等分试样的DNA。这显示了通过Reb1-MNase融合进行基因组DNA的逐步消化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55836/55836fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55836/55836fig3.jpg" /> 图3 :Reb1-MNase和游离MNase ChEC-seq实验的基因组浏览器快照。加入Reb1的片段末端信号和加钙后游离的MNase ChEC-seq 30s的IGV视图和Med8和游离的MNase ChEC-seq 1分钟af<html>沿着酵母基因组的代表性部分添加钙。通过将映射到每个基本位置的片段结束数除以映射的片段结束总数乘以映射的总碱基数来对数据集进行归一化。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55836/55836fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55836/55836fig4.jpg" /> 图4 :Reb1-MNase释放的片段在Reb1有机网站周围的丰富。通过ORGANIC 13测定的大约1,991个Reb1基序的30s Reb1和游离MNase ChEC-seq片段末端信号的平均图。数据如图3所示归一化。请点击这里查看该图的较大版本。 <table fo:keep-together.within-page="1"><tbody><tr><td> 质粒 </td><td> 酵母选择标记 </td><td> 笔记 </td><td> Addgene质粒编号 </td></tr><tr><td> pGZ108 </td><td> kanMX6 </td><td> 3xFLAG-MNase标记，33aa接头</td><td> 70231 </td></tr><tr><td> pGZ109 </td><td> HIS3MX6 </td><td> 3xFLAG-MNase标记，33aa接头</td><td> 70232 </td></tr><tr><td> pGZ110 </td><td> TRP1 </td><td> 3xFLAG-MNase标记，33aa接头</td><td> 70233 </td></tr><tr><td> pGZ136 </td><td> URA3 </td><td>表达在REB1启动子控制下的3xFLAG-MNase-SV40 NLS </td><td> 72273 </td></tr><tr><td> pGZ173 </td><td> kanMX6 </td><td> MNase标签，8 aa连接子</td><td> 70234 </td></tr></tbody></table>表1:ChEC质粒的细节。所有标记载体均基于pFA6a载体，因此与常用的F2 / R1标签引物对相容。标记盒由指定长度的接头组成，3xFLAG表位，以便于通过蛋白质印迹检测（除了在pGZ173的情况下，缩短了接头以除去3xFLAG标签），MNase的成熟链（GenBank P00644 ，aa 83-231）和所示的可选标记。 <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> [最后] </td></tr><tr><td> 5倍PCR缓冲液</td><td> 10 mL </td><td> 1x（2mM MgCl 2 ） </td></tr><tr><td> 10mM dNTP混合物（每个dNTP 2.5mM） </td><td> 1 mL </td><td> 200mM（每个dNTP 50mM） </td></tr><tr><td> 10mM F2引物</td><td> 2.5 mL </td><td> 0.5 mM </td></tr><tr><td> 10 mM R1 p里默</td><td> 2.5 mL </td><td> 0.5 mM </td></tr><tr><td> pGZ108 / 109/110/172（1-5ng / mL） </td><td> 1 mL </td><td></td></tr><tr><td> 2 U /μL热启动高保真聚合酶</td><td> 0.5 mL </td><td> 1 U </td></tr><tr><td> ddH 2 O </td><td> 32.5mL </td><td></td></tr></tbody></table> 表2:用于PCR扩增3xFLAG-MNase标记盒的反应混合物。 F2 / R1引物序列可以在http://yeastgfp.yeastgenome.org/yeastGFPOligoSequence.txt找到。 <table fo:keep-together.within-page="1"><tbody><tr><td> 温度（°C） </td><td> 时间 </td><td> 周期 </td></tr><tr><td> 98 </td><td> 30秒</td><td> 1 </td></tr><tr><td> 98 </td><td> 10秒</td><td> 25 </td></tr><tr><td> 55 </td><td> 30秒</td><td> 25 </td></tr><tr><td> 72 </td&gt; <td> 1分15秒</td><td> 25 </td></tr><tr><td> 72 </td><td> 2分钟</td><td> 1 </td></tr><tr><td> 10 </td><td>永远</td><td>保持</td></tr></tbody></table> 表3:3xFLAG-MNase标记盒的PCR扩增的热循环条件。孵育温度和延长时间可能需要根据所使用的DNA聚合酶进行调整。 <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> [最后] </td></tr><tr><td> 10x Taq缓冲液</td><td> 5 mL </td><td> 1x（1.5mM MgCl 2 ） </td></tr><tr><td> 10mM dNTP混合物（每个dNTP 2.5mM） </td><td> 1 mL </td><td> 200mM（每个dNTP 50mM） </td></tr><tr><td> 10 mM检查底漆</td><td> 1 mL </td><td> 0.2mM </td></tr><tr><td> 10mM MNase-R或阴性对照引物</td><td> 1 mL </td><td> 0.2mM </td></tr><tr><td> 5U / mL Taq聚合酶</td><td> 0.5 mL </td><td> 2.5 U </td></tr><tr><td> ddH 2 O </td><td> 41.5mL </td><td></td></tr></tbody></table> 表4:用于菌落PCR确认MNase标记的反应混合物。检查引物序列可以在http://yeastgfp.yeastgenome.org/yeastGFPOligoSequence.txt找到。将检查引物用作MNase（5&#39;-TTGTGCAGCTTCTTGGTAC-3&#39;）内的反向引物的正向引物和作为阴性对照的各基因下游的反向引物〜500碱基对（bp）。 <table fo:keep-together.within-page="1"><tbody><tr><td> 温度（°C） </td><td> 时间 </td><td> 周期 </td></tr><tr><td> 95 </td><td> 5分钟</td><td> 1 </td></tr><tr><td> 95 </td><td> 30秒</td><td> 35 </TD&gt; </tr><tr><td> 55 </td><td> 30秒</td><td> 35 </td></tr><tr><td> 68 </td><td> 1分钟</td><td> 35 </td></tr><tr><td> 68 </td><td> 5分钟</td><td> 1 </td></tr><tr><td> 10 </td><td>永远</td><td>保持</td></tr></tbody></table> 表5:菌落PCR确定MNase标记的热循环条件。孵育温度和延长时间可能需要根据所使用的DNA聚合酶进行调整。 <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> [最后] </td></tr><tr><td> 1M Tris，pH7.5 </td><td> 1.5 mL </td><td> 15 mM </td></tr><tr><td> 1 M KCl </td><td> 8 mL </td><td> 80 mM </td></tr><tr><td> 0.2 M EGTA </td><td> 50 mL </td><td> 0.1mM </td></tr><tr><td> ddH 2 O </td><td>填100毫升</td><td></td></tr></tbody></table> 表6:缓冲液A的配方在使用前，加入一半的蛋白酶抑制剂片剂，50μL的100mM PMSF（最终1mM），5μL的200mM精胺（最终0.2mM）和2.5μL的1M亚精胺（0.5mM最终）/ 5mL溶液。 <table fo:keep-together.within-page="1"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> [最后] </td></tr><tr><td> 5 M NaCl </td><td> 8 mL </td><td> 400 mM </td></tr><tr><td> 0.5M EDTA </td><td> 4 mL </td><td> 20 mM </td></tr><tr><td> 0.2 M EGTA </td><td> 2 mL </td><td> 4 mM </td></tr><tr><td> ddH 2 O </td><td>填充至100 mL </td><td></td></tr></tbody></table> 表7:2x停止溶液的配方。合并90μL的stop溶液与10μL的10％SDS在微量离心管中，每个时间点（参见步骤2.4）。

Watch this Scientific Journal Video about Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae at JoVE.com

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

我们描述了染色质内源性切割与高通量测序（ChEC-seq），染色质免疫沉淀（ChIP） - 正交法，用于在全基因组范围内用微球菌核酸酶（MNase）融合蛋白测定蛋白质结合位点。

与ChEC-seq的蛋白质-DNA相互作用的全基因组映射<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

genome-wide-mapping-protein-dna-interactions-with-chec-seq

Nanyang Technological University

Research

JoVE Journal

Genetics

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

11.0K 次观看.  Fred Hutchinson Cancer Research Center. 我们描述了染色质内源性切割与高通量测序（ChEC-seq），染色质免疫沉淀（ChIP） - 正交法，用于在全基因组范围内用微球菌核酸酶（MNase）融合蛋白测定蛋白质结合位点。 

视频: Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

ATAC 基因组在原发性人类 T 淋巴细胞中的染色体全易接近染色质

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

增殖和静止人成纤维细胞的全基因组转录衰变率的测定

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

从酿酒酵母中组蛋白修饰的染色质沉淀 (芯片)

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

启动子捕获高分辨率, 全基因组的启动子交互分析

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

酿酒酵母4-硫尿嘧啶代谢标记及新合成 mRNA 作为 RNA 聚合酶 II. 活性的一种代理

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...